The long term goal of this project is to understand the molecular basis of the biological properties of the murine coronavirus, mouse hepatitis virus strain A59 (MHV-A59). MHV-A59 causes persistent demyelinating disease in the central nervous system of mice and readily establishes chronic, productive, non-lytic infections in glial cells in culture. Thus, the virus presents a model system in which to study viral persistence, tissue tropism and virus- induced demyelination. MHV infection of mice has been cited as a model for Multiple Sclerosis. We have been engaged in a detailed analysis of the molecular biology of MHV-A59. During the next five year period studies on the structure and function of the MHV-A59 non-structural genes, in particular the viral RNA-dependent RNA polymerase and the mechanism of transcription of mRNAs will be carried out. Antisera raised against bacterial/viral fusion proteins derived from cloned cDNA fragments from non-structural genes A,B,D, and E of MHV-A59 will be used to identify the proteins encoded by these genes. A map of the polypeptides encoded by the 24 kb putative polymerase gene A as well as the complete nucleotide sequence of the gene will be generated. As a long term goal the antisera will be used in an in vitro system to define the individual activities of the polymerase polypeptides. Studies will be carried out to test the leader-priming model for the initiation of viral mRNA transcription. Transfection of RNAs transcribed from pGEM vectors containing viral intergenic sequences upstream of the CAT gene will be carried out to determine whether these viral sequences can function as promoters for transcription by a leader primed mechanism. Site specific mutagenesis of leader and intergenic regions will identify nucleotide critical for transcription. In addition, RNA binding proteins will be identified by gel retardation assays and by assaying specific retention of leader intergenic RNA on protein blots. A long term goal will be to assemble a complete cDNA copy of the genome to be expressed as RNA to be used for mutagenesis to map the pathogenic properties of the virus.